ELECTROLYTE REQUIREMENTS OF PROTISTS AND ARCHEOMETABOLISM

By S. H. Hutner, Marvin Sanders, J. J. A. McLaughlin, and Stanley Scher Haskins Laboratories, New York, N . Y.

Rubey reasons that the ocean was salty and substantially unchanged through- out geological time and that the increment of salts deposited by leaching of the land was counterbalanced by an increase in the amount of water.’ Conse- quently, if we assume that life began in the sea, electrolytes were very much a part of the environment for primordial life. The occurrence of inorganic elec- trolytes as essential constituents of organisms might be construed as the in- delible imprint of the sea, as Macallum2 regarded the pattern of salts in the body fluids of metazoa. Unfortunately, we are densely ignorant when we con- sider the question: to which part of the cell are electrolytes indispensable?

Metazoan metabolism supplies further incentives for work on the funda- mentals of electrolyte requirements. hfovements of Na+ and K+ characterize nervous activity ;3 what mode of intracellular communication preceded this development? To trace the physical basis of thought to its beginnings ob- viously requires a stupendous knowledge of the nature of life; less ambitiously, it demands an understanding of the mode of action of hormones. Certain steroid hormones drastically influence electrolyte relations in vertebrates; where‘are the protistan forerunners-the Anlugen-of these systems?

Our bias, directed by drudgery in the pursuit of indispensable trace elements and growth factors that lie well beyond the limits of sensitivity of conventional chemical analysis, makes us wonder at the current preoccupation with those quantitatively most abundant linear macromolecules, the nucleic acids and fibrous proteins that are invariant cell constituents, and with such conspicuous molecules as adenosine triphosphate (ATP). Investigation of the role of quantitatively minute invariants can be postponed only a t the peril of impeding an increase in our knowledge of these macroconstituents. We are inclined to look upon certain inorganic electrolytes as indispensable to life and as capable of revealing much about its nature. While nucleic acids and associated protein may well be the repositories of genetic information, how is this information communicated to the effectors of the cell? We find it difficult to conceive of genetic information as merely a punched tape. The means of communication are part of this information, and the problem of communication is inseparable from that of the storage and duplication of information. In short, experiments designed to trace the ancestry of neurohumoral mechanisms, in which electro- lytes play an important role, should bring one close to what the French call the “secret of life.”

Some components of the original cell machine must have composed the sub- stratum for the evolution of the neurohumoral systems that underlie thought, as closely as any reaction chains can be said to do this. The narrower problem considered here is: Can the electrolyte requirements of extant organisms pro- vide clues to the origin of these systems?

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Hutner et al. : Electrolyte Requirements of Protists 28 7 It is not known how a hormone functions a t the cellular correspond-

ingly, we have almost no information on the equivalent in protists of the neuro- chemical systems in metazoa. Clearly, new research tools are needed, and we believe that protists might serve this purpose.

Before investing effort in elucidating the mode of action of electrolytes, en- visioning them as archaic prerequisites for life, it is well to be assured that their assumed fundamental essentiality is borne out by their being universal nutri- tional requirements, apart from the gross use of electrolytes as osmoregulatory agents. The picture is reassuring. First, there is the universality of the K and Mg requirements.

Fresh-water blue-green algae need Na and K;6 there can be little doubt that the blue-greens are a very conservative group. I t is not known whether land bacteria require Na or halogens. Since autoclavable polyethylene culture vessels and chemical equipment are becoming available, it should soon be prac- ticable to purify nutrient chemicals adqeuately ; electrolytes are leached out of glass too easily. The sporadic occurrence of chlorine in antibiotics of bacterial origin (chlortetracycline and chloramphenicol, for example) hints that chlorine is essential for bacteria. This element is an essential micronutrient for higher plants.? The many reports of the use of NaCl as a fertilizer favor the likeli- hood that Na also will prove to be essential. Nothing is known about electro- lyte requirements in fresh-water or soil protists other than bacteria.

The “why” of salt requirements in vertebrates is almost wholly obscure, aside from the connection of salt with nerve conduction and certain steroid hormones, and the occurrence of HCI in gastric Thanks to our knowledge bf thy- roxine, the essentiality of iodine for vertebrates is recognized; the sporadic occurrence of iodotyrosine compounds in sponges and coelenterates might other- wise be dismissed as a biochemical freak. Perhaps the situation is similar for bromine, which was first known biologically in the molluscan product Tyrian purple (a brom-indigo); bromine may be an essential for vertebrate^.^, l o

I t is good strategy to study a function in a situation in which it is displayed in a specialized way on a large scale. To study locomotion, for example, one investigates muscle, not amoeba; to study fermentation, one investigates yeast. Which organisms are best for work on an electrolyte? Marine algae and pro- tozoa have conspicuous salt requirements; some of the strains in pure culture seem to be exceptionally favorable material. One of the extreme forms is the green brine-flagellate Dunaliella salina, which can grow in tenfoId concentrated sea-water or saturated NaCl.”

Some years ago we attempted to learn whether the tetraethylammonium ion spared the Na requirement of Dunaliella; we were inspired by the reportI3 that the conductivity of certain frog nerves, placed in Na-free solutions, was restored by tetraethylammonium. The results were inconclusive. However, these experiments did show us that there was need for a metabolically inert, pure, low-molecular nonelectrolyte to serve to satisfy any nonspecific osmotic requirements. hletabolically inert, low-molecular cationic, anionic, and zwit- terionic electrolytes were also required to satisfy any nonspecific need for an electrolyte and, for a brine alga and a salt-requiring bacterium, these spared Na. Our results also indicated that while triethanolamine and tetraethyl-

288 Annals New York Academy of Sciences ammonium chloride did not spare Na for Dunaliella, the presence of penta- erythritol, as a presumable inert means of supplying additional osmotic pres- sure, reduced the Na requirement to 10 per cent of the minimal requirement in our standard medium; that is, from 5 per cent to 0.5 per cent. For some reason sugars inhibited growth. It was reported recently that the conduc- tivity of another type of frog nerve kept in a Na-free solution was restored by guanidine ch10ride.l~ This has special interest, because a guanidine-like ion may be responsible for the maintenance of irritability in Mitella cells washed with distilled water.14 Consequently, we have formulated a new working hypothesis; namely, that the replacement or sparing of Na (and perhaps K) may require a combination of organic cations. A number of years ago, in col- laboration with Herman Ziffer of Mount Sinai Hospital, New Pork, N. Y., we attempted to ascertain whether the heightened need for Na in adrenalectomized rats could be met, at least in part, with such bulky, strong organic bases as tetraethylammonium but, owing to a succession of mischances, the experiments were inconclusive. We are unaware of any published work describing such at tempts.

Much information on the mode of action of vitamins has come from studies on compounds that spare or replace them. If a vitamin participates in syn- thesis, and an organism that needs an exogenous source of this vitamin is sup- plied with the finished products of these syntheses, the vitamin may, in effect, be bypassed. The search for substitutes for the NaCl requirement of Duna- liella may be similar in principle. I t is conceivable that, as organisms evolved, they elaborated organic substitutes for inorganic electrolytes. Where this seems to have happened, the osmoregulatory effect of electrolytes appears to be the function concerned. Thus, sharks use urea to maintain the isotonicity of their blood with sea water, and perhaps crustaceans use amino acids for the same This phenomenon suggests that part of the difficulty in demonstrating electrolyte requirements in nonmarine organisms may stem from the evolutionary development of electrolyte-bypassing factors as part of the evolution toward the fixity of the milieu intirieur, and that a trace requirement for electrolyte represents the least easily bypassable, the most indispensable- in short, the most primitivefunction of an electrolyte. At this point, where the problem is to demonstrate trace requirements, one might invoke another principle that originated in microbiology and that has been widely applied in biochemistry; namely, the use of inhibitors to increase the need for a metabolite and thus make it easier to demonstrate. The sulfa drugs provide the classic example. They were used first to demonstrate that p-aminobenzoic acid was a metabolite, and then in the demonstration that vitamin BIZ was a product of a synthetic system in which p-aminobenzoic acid played a part. There are a few indications in the literature that this technique may be applied to the study of electrolyte requirements. N-Amylcarbamate is competitive with Na in constant ratio for the maintenance of the conductivity of nerve tissue.I7

Competition with Na by derivatives of guanidine was noted by Larramendi, Lorente de N6, and Vidal.I3 We have encountered exaggeration of the K re- quirement of bacteria by triethanolaminel* in unpublished work with bacilli and algae. No reports have yet appeared on whether this holds true for pro-

Hutner et al. : Electrolyte Requirements of Protists 289 tozoa and metazoa. The report that 2,4-diaminobutyric acid acts as a com- petitor for K in tissues1g seems not to have been applied to protists as yet. Competition between K and Na is well known.

There may be an exception to our general ignorance of the action of hormones a t the cellular level-if a yeast “cell” is functionally akin to a metazoan cell. Conway202 reports that deoxycortisone inhibits the uptake of K and the release of Na by yeast so grown that it had accumulated Na, and that compound E stimulates the uptake of K by yeast. This might be the basis of a microbio- logical test for hormones that influence electrolytes. Since animals that are deficient in corticoid hormones must be given extra Na, Na-retaining hormones are, in effect, Na-sparing factors. Steroid requirements are not uncommon in protists, for none is a steroid hormone active. Indeed, it is not even known whether steroid hormones occur in protists. Conway’s observation thus is an isolated one; its extension to other hormones and protists might support his idea that regulation of permeability was the original function of hormones.

To investigate any possible relation between the electrolyte requirements of marine organisms and the postulated archaic, persistent, and unknown func- tions of electrolytes in all present organisms is to raise the question of which marine organisms to study, keeping in mind that lipid-soluble nutrients such as steroids must be taken into account. There is no evidence that brine flagellates such as Dunaliellu salina are permeable to steroids. The probability that they are so seems slight, because Dunaliellu so far has resisted all efforts to grow it in the dark. This may indicate an impermeability to energy-fur- nishing nutrients and, perhaps, to most other organic nutrients as well. There are marine protists that are able to absorb water-insoluble materials; these are the particle-ingesting (phagotrophic) protozoa. Many of these are photo- synthetic flagellates. One such fresh-water flagellate, Ochromonas malhamen- sis, is used widely in the assay of vitamin B12, because the specificity of its requirement of this nutrient is identical, by present indications, with that of vertebrates;20 it seems to match the chick in ability to utilize vitamin BIZ in crude natural materia1s;l perhaps because of its phagotrophy. For this and other reasons of comparative biochemistry, flagellates such as chrysomonads might be closer to the metozoa than any other protists now available in pure culture, and they might be favorable tools for use in the effort to find Anlagen of metazoan hormones. Certainly, a protist of this kind would be needed if electrolyte-sparing factors were bulky, lipid-soluble, or poorly soluble in aque- ous and nonaqueous solvents alike. Since 0. malhamensis is a fresh-water form, its requirements for monovalent electrolytes other than K may be minute and demonstrable only with the combined aid of rigorously purified chemicals and the aforementioned antagonists of electrolytes.

The bromine require- ment in mice and chicks noted previouslyg- lo was brought to light by theanaly- sis of the nutritional supplements that were required to overcome the depres- sion of growth caused by feeding thyroactive materials. We have been studying the greatly enhanced nutritional requirements of 0. malhamensis grown a t incubator temperatures above the usual optimum. We are looking for parallels with the fever-heightened nutritional requirements in metazoa. Heightened

Another approach to this problem has been made.

290 Annals New York Academy of Sciences temperature, in fraying the biochemical fabric of the organism, brings to light otherwise poorly accessible metabolic chains. The appearance of electrolyte requirements as “temperature factors” would support the idea that bromine and other characteristically marine, but biochemically unfamiliar, electrolytes are prerequisites for life. The effectiveness of the temperature approach may be gauged from the fact that the vitamin B I ~ requirement of 0. malhamensis is so augmented by elevated temperature (- five hundredfold and, in animals fed thyroactive materials, - fourfold) that, had vitamin BIZ and cobalt been unknown as growth factors, they easily could have been identified in the role of “temperature factors.” These results are being prepared for publication.

Marine phagotrophic chyrsomonads are common, but none are yet in pure culture. However, the increasing success in cultivating delicate marine dino- flagellates, diatoms, obligately photosynthetic chrysomonads, and several other kinds of marine plankton protistsz2, encourages the hope that the cultivation of highly phagotrophic marine chrysomonads may be achieved soon. These plants might well be the most favorable organisms of all for studying the na- ture of electrolyte requirements.

Before ending this account of protistan tools, present and potential, for elec- trolyte studies, mention should be made of the red halophiles, which are a group of organisms similar to the myxobacteria. Most of them need a t least 14 per cent NaCl to grow. The sliminess of these extreme halophiles and of several colorless halophilic bacteria resides in an extracellular layer of deoxyribonu- clease (DNA), as shown by direct isolation and removal of the slime layer with

One is tempted, on the basis of the DNA layer of halophiles and the extreme sensitivity of DNA to variations in ionic strength, to ascribe to electrolytes a role in controlling the ionization and degree of hydrogen bonding of DNA.26 Perhaps the specificity of electrolytes originally depended on different spatial constraints along the closely packed DNA helix and its associated protein. The DNA layer of the halophiles suggests that the converse may hold, and that DNA originally controlled directly the water and electrolyte equilibrium of the organism. Intimacy with the environment could hardly go further; no func- tion save that of reproduction itself could be more vital. Much of evolution, then, might have been, and still can be, the elaboration of controls interposed between DNA and agents for homeostatic responses to the environment. DNA was thus protected, ever more effectively, from fluctuations in the environ- ment, which was all the better for genetic stability. The physical state of extracted DNA (a polyelectrolyte) is highly sensitive to variations in the ionic strength of the medium. NaCl, for example, protects it against heat denatura- tion, and divalent cations such as Ca++ and Mg++ stabilize it much more effi- ciently than do monovalent Exactly how electrolytes affect the shape of DNA and vital proteins (and thus, necessarily, their function) presents a problem for future investigation. The present-day electrolyte requirements are so many clues, could we but read them, to the primordial functions of DNA or whatever substance served as the original center of information and com- munication.

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